Gene recombination technology, which has been recently developed with a great advance, allows the mass production of the proteins which are derived from higher organisms by introducing the genes of interest into microorganisms. Largely, of interest are the proteins that are medicinally useful because they are of high value. Demand for proteinaceous medicines of high purity is expected to increase explosively as there continue to be discovered diseases that are intractable, but curable with such proteinaceous medicines. Thus, there are needed techniques in which functional recombinant proteins can be produced at relatively low costs through various microorganisms harmless to the body.
Yeast, a microorganism which performs protein expression and secretion like an eucaryote, is usefully utilized as a host through which recombinant proteins derived from higher organisms can be produced on a large scale. Typically, Saccharomyces cerevisiae is used as such a host in the study on recombinant protein production using yeast. However, the strain is now regarded unsuitable in the following aspects: recombinant proteins are produced in low yields on account of not only the absence of a strong promoter for the effective expression of exogenous proteins, but also the instability of the plasmids introduced into the yeast upon long-term fermentation; there is needed fed-batch fermentation when the strain is cultured at a high concentration; and expressed exogenous proteins undergo hyperglycosylation (Romanos, et al., Yeast, 8: 423 (1992)). An exogenous protein expression system to overcome the above problems was developed in Pichia pastoris, a methanol-assimilating yeast (Sudbery et al., Yeast, 10: 1707 (1994); Cregg et al., Bio/Technol. 5: 479 (1987)). In addition, active research has been directed to the development of exogenous protein expression systems using Hansenula polymorpha, a methanol assimilating yeast (Gellissen et al., Bio/Technol. 9: 291 (1991); Janowicz et al., Yeast 7: 431 (1991)). Hansenula polymorpha, which is gathering strength as a novel host cell for producing recombinant proteins, utilizes methanol as a carbon source and thus, can be mass-cultured with ease. In addition, this yeast strain contains a strong promoter for several genes relevant to its methanol metabolism and allows the multicopy integration of exogenous genes into its genomic DNA so that the plasmids can be stably maintained even when it is cultured at high concentrations.
In the case that recombinant proteins are produced by use of yeast, not only is an effective expression and secretion system necessary for the enhancement of the yield, but it is very important to prevent proteinases from degrading the exogenous proteins expressed and secreted. Usually, the culture of recombinant yeasts for a long period of time in a fermentation bath suffers from a problem in that proteinases secreted from the host cells to the media naturally or through cell lysis degrade the produced recombinant proteins to lower the production yield of the recombinant proteins. In fact, analysis through, for example, HPLC and MS demonstrated that a substantial part of the recombinant proteins, such as human epidermal growth factor secreted from recombinant Saccharomyces cerevisiae(George-Nascimento et al., Biochemistry 27:797(1988)) and Pichia pastoris(Clare et al., Gene 105:205(1991)) cells to their culture media were degraded at their carboxyl ends. It was postulated that carboxypeptidases of the host cells removed one or two amino acids from the carboxyl ends of the recombinant proteins secreted.
Corresponding to the lysosomes of higher cells, the vacuoles of Saccharomyces cerevisiae contain various proteinases and are responsible for proteolysis upon depletion of nutrition. Particularly, carboxypeptidase Y is utilized for the carboxyl-terminal amino acid analysis by virtue of its capacity of hydrolyzing various protein substrates and is a model protein under active and extensive study on protein sorting and targeting (Rothman et al., Cell, 47: 1041 (1986); Johnson et al., Cell, 48: 875 (1987); Valls et al., J. Cell. Biol., 111:361 (1992)). In addition, the carboxyl-terminal degradation which takes place upon the over-expression of exogenous proteins is also known to be due to carboxypeptidase Y. Carboxypeptidase Y genes are reported to be cloned from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Schizosaccharomyces pombe (Valls et al., Cell, 48: 887 (1987); Mukhtar et al., Gene, 121: 173 (1992); Ohi et al., Yeast, 12: 31 (1996); Tabuchi et al., J. Bacteriol., 179: 4179 (1997)).
Along with carboxypeptidase Y, Saccharomyces cerevisiae protease A is present within vacuoles, playing a role in hydrolyzing proteins. Further, protease A takes part in the proteolytic process of vacuolar proteases, such as protease B, carboxypeptidase Y and aminopeptidase Y, as well as vacuolar hydrolases, such as RNase, alkaline phosphatase, and acid trehalase (H. B. Van Den Hasel et al., Yeast, 12: 1 (1996)). Particularly in the strains whose gene PEP4 is disrupted, the activity of carboxypeptidase Y is significantly reduced. Accordingly, since the activity of carboxypeptidase Y is significantly lowered in a Hansenula polymorpha whose PEP4 gene is disrupted, the disruption of the PEP4 gene on the genome can make various lyases, including carboxypeptidase Y, low in enzymatic activity. Gene PEP4 is cloned from Saccharomyces cerevisiae, Candida albicans, and Neurospora crassa as disclosed in several documents (Ammerer et al., Mol, Cell. Biol., 6: 2490 (1986); Woolford et al., Mol. Cell. Biol., 6: 25 (1986); Lott et al., Nucleic Acids Res., 17: 1776 (1986); Bowman et al., Genbank Accession No U36471).
The gene KEX1 of yeast is known to code for carboxypeptidase a that is involved in the processing of killer toxins K1 and K2 and an α-factor (mating pheromone) precursor (Alexander et al., Cell, 50: 573 (1987)). Carboxypeptidase α is a digestive enzyme that hydrolyzes the carboxyl-terminal peptide bond in polypeptide chains. Hydrolysis had been known to occur most specifically if the carboxyl-terminal residue is a basic amino acid such as arginine or Ivsine. However, expression of hirudin, a thrombin inhibitor, in Saccharomyces cerevisiae demonstrated that the specificity of carboxypeptidase α is not confined to basic amino acids, but extended further to non-basic amino acid, such as tyrosine, leucine and glutamine, at the carboxyl end (Hinnen et al., Gene Expression in Recombinant Microorganism, 155: 164 (1994)).
Expression systems for Pichia pastoris in use were usually developed by introducing in the microorganism truncated expression vectors which were then allowed to be inserted at the site of gene AOX1 or HIS4 through homologous recombination. When an expression cassette composed of an AOX1 promoter and a terminator is inserted to the site of gene AOX1, disruption occurs in the gene AOX1, creating an aox1 transformant. While the normal strain produces a large quantity of AOX1 enzyme upon methanol culture, the aox1 strain cannot produce the AOX1 enzyme any more, exhibiting a very slow growth rate (methanol utilization slow: Mud). Hence, this mutant has an advantage over the AOX1 wild type (Mut+) in that the mutant can be grown in an even sparser oxygen atmosphere than the wild type can. There are several reports which reveal the superiority of the MutS recombinant strain to the Mut+ strain in recombinant protein production yield through the fermentation by use of the MutS recombinant strain and the Mut+ strain, indicating that the MutS strain is more useful for the mass production of some recombinant protein (Cregg et al., Bio/Technology 5: 479 (1987); Romanos et al., Vaccine 9: 901 (1991)).
In contrast, conventional expression systems for Hansenula polymorpha were developed by taking advantage of the phenomenon that a multicopy of an exogenous gene is tandemly introduced to non-specific sites of the genome. Accordingly, intact expression vectors, which are not cut, but circular, are introduced into the host (Janowicz et al., Yeast, 7: 431 (1991); Gelalissen et al., Trends Biotechnol. 10: 413 (1992); Gatzke et al., Appl. Microbiol. Biotechnol. 43: 844 (1995)). In this case, the conventional expression systems suffer from a significant problem in that, because different expression efficiencies appear depending on the host genome sites to which the expression vectors are inserted, there is needed the consumptive searching procedure of analyzing expression yields of numerous transformants to select the transformant which is the most productive of the recombinant protein of interest. In addition, unlike the Pichia pastoris expression system which is high in homologous recombination frequency, Hansenula polymorpha systems, even though utilizing an MOX promoter and an MOX terminator, make exogenous genes inserted, for the most part, to non-specific sites of the host genome. Further, even when the exogenous gene is inserted to the MOX gene site at a low frequency, the vector is incorporated as being intact, so that the MOX genes of the transformants are not damaged. In methanol culture media, these MOX transformants, to experimenters' disappointment, show poorer expression yields for the recombinant protein of interest than expected because most of the methanol fed is consumed as the substrate of the MOX enzyme which is of high activity (Kim et al., Biotechnol Lett. 18:417 (1996)). In the MOX wild types cultured in methanol, moreover, the expressed MOX protein amounts to as much as 30-40% of the total expressed proteins (Guiseppin et al., Biotechnol. Bioeng. 32:577 (1988)), resulting in relatively reducing the expression efficiency of the recombinant protein of interest.
For Hansenula polymorpha, there have been not yet developed techniques by which expression cassettes inserted in the host genome can be rendered to pop out later. Thence, as indicated in the report of Hodgkins et al. (Hodgkins et al., Yeast 9:625), even after a desired mutant is obtained by using as a mother strain a transformant carrying an expression cassette for a particular recombinant protein, the mutant, which is obtained under difficulties, cannot be used as a general host to express various recombinant proteins because of the incapability of popping out the preexisting expression vector from the host genome and thus of introducing a new expression cassette into the host genome.